Structure, function, and disease biology of MICU1/MICU2

摘要 线粒体通过钙激活通道（称为单向转运体）摄取钙。钙的摄取使得 细胞器的代谢状态与快速变化的能量需求相匹配，反过来， 神经传递和肌肉收缩等关键过程。单向转运蛋白是钙激活的钙 由钙结合异源二聚体MICU 1/MICU 2调节的通道。MICU 1的突变最近被 被确定为一种新形式的肌病的原因，其特征是疲劳和运动不耐受， 线粒体肌病的典型特征MICU 1和MICU 2感知的精确机制 钙调节单向转运体，以及这种异源二聚体的病变如何导致这种极不寻常的肌病， 不知道。通过这一双重PI赠款，我们建议：（1）表征的物理化学性质， MICU 1和MICU 2。野生型蛋白和突变体在大肠杆菌中表达。大肠杆菌的特征在于 寡聚状态、结构稳定性、Ca 2+和Mg 2+结合亲和力和pH敏感性。(2)确定 MICU 1和MICU 2调节单向转运蛋白的结构基础。高分辨率X射线结构 将测定单独的MICU 2以及载脂蛋白和Ca 2+结合形式的MICU 1/2异二聚体。 电子冷冻显微镜将用于确定MICU 1/2的天然样寡聚体的结构 复杂. (3)研究细胞系统中的线粒体钙动力学。利用基因组编辑技术 我们已经设计了一个强大的体内敲除细胞系系统，用于研究工程和 MICU 1和MICU 2中天然存在的人类突变对线粒体钙转运动力学的影响， 能量学，以及（4）通过以下方式了解人类MICU 1肌病的代谢和生物能量基础： 研究Micu 1-/-小鼠作为模型。我们将描述肌肉组织学，线粒体 生物能量学、运动表现和代谢组学以及单纤维收缩性来检验以下假设： MICU 1的缺失通过引起能量代谢紊乱而导致肌病。我们的目标-跨越 从分子到动物生理学--将产生对调节的整体和机械的理解， MICU 1和MICU 2的线粒体钙摄取及其对新描述的人类 肌病

ABSTRACT Mitochondria uptake calcium via a calcium activated channel called the uniporter. Calcium uptake allows the organelle's metabolic state to be matched to rapidly changing energy requirements, and, in turn, tune key processes such as neurotransmission and muscle contraction. The uniporter is calcium-activated calcium channel regulated by the calcium binding heterodimer MICU1/MICU2. Mutations in MICU1 have recently been identified as a cause of a new form of a myopathy characterized by fatigue and exercise intolerance without the classical features of mitochondrial myopathy. The precise mechanisms by which MICU1 and MICU2 sense calcium to regulate the uniporter, and how lesions in this heterodimer lead to this highly unusual myopathy are not known. Through this dual PI grant we propose to: (1) Characterize the physicochemical properties of MICU1 and MICU2. The wild type proteins and mutants expressed in E. coli will be characterized with respect to the oligomeric state, structural stability, Ca2+ and Mg2+ binding affinities, and pH sensitivity. (2) Determine the structural basis for the regulation of the uniporter by MICU1 and MICU2. High resolution X-ray structures of MICU2 alone and of the MICU1/2 heterodimer in the apo and Ca2+-bound forms will be determined. Electron cryo-microscopy will be used to determine the structure of a native-like oligomer of MICU1/2 complex. (3) Investigate mitochondrial calcium dynamics in cellular systems. Using genome-editing technology we have engineered a powerful in vivo system of knockout cell lines for studying the effects of engineered and naturally occurring human mutations in MICU1 and MICU2 on the mitochondrial calcium transport kinetics and energetics, and (4) Understand the metabolic and bioenergetic basis of human MICU1 myopathy by investigating the Micu1-/- mouse as a model. We will characterize the muscle histology, mitochondrial bioenergetics, exercise performance and metabolomics, and single fiber contractility to test the hypothesis that loss of MICU1 leads to a myopathy by causing disturbances in energy metabolism. Our aims – spanning the molecular to animal physiology -- will yield a holistic and mechanistic understanding of the regulation of mitochondrial calcium uptake by MICU1 and MICU2 and its contribution to a newly described human myopathy.